We have previously shown that glutithione (GSH) increases the internalization ant T-cell proliferative activity of interleukin-2 (IL-2). Here we examine whether GSH may regulate the cytolytic activity of cytotoxic T cells through its effect on IL-2. We generated anti-CD3-activated killer cells (CD3-AK) which were exclusively CD3+ and CD8+ cells. The cytolytic activity of these killer cells against a tumor target cell (HFL/b) was dependent on the epresence of GSH and IL-2 or T-cell mitogens such as phorbol ester (e.g., PMA). The PMA-induced CD3-AK killing was IL-2 dependent and was inhibited by anti-IL-2 antibodies. This cytolytic activity of CD3-AK was abolished by depletion of cellular GSH with L-buthionine-(S,R)-sulfoximine (BSO; 0.05-0.5 mM for 10-24 h), an inhibitor of de novo GSH synthesis. The inhibitory effect of BSO (0.05 mM) was reversed by the addition of GSH. In the absence of IL-2 or PMA, GSH alone did not induce the cytolytic activity of CD3-AK. These results together with our previous finding of GSH effect on the internalization of IL-2 suggest that intracellular GSH may regulate the effect of IL-2 and affect the cytolytic activity of IL-2 dependent cytotoxic T-cells.